GSM2537915: ChIPseq NC NELF; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
NELFA
Cell type
Cell type Class
Breast
Cell type
Breast cells
NA
NA
Attributes by original data submitter
Sample
source_name
Normal breast tissue, unsorted, non-carrier, NELF-A ChIP
genotype/variation
Non-Carrier
tissue
Normal breast
sorted cell type
Unsorted
antibody
anti-NELF-A
individual identifier
BSC132
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
DRIP-seq: DRIP assay was performed following the established protocol. Briefly, cells were washed twice in PBS, and resuspended in TE (Sigma, T9285) containing 5μl of proteinase K (Roche, 03115828001) and a final concentration of 0.5% SDS and proteinase K (Roche, 03115828001). Samples were incubated overnight at 37C. Genomic DNA was extracted using phenol/chloroform (Sigma, P2069) in phase lock tubes (5PRIME, 2302840) and ethanol precipitated. DNA was digested using established restriction enzyme cocktail (HindIII, EcoRI, BsrGI, XbaI and SspI) overnight at 37C. Digested DNA was cleaned up by phenol-chloroform extraction and ethanol precipitation. For DRIP, digested DNA was incubated with S9.6 antibody (Kerafast, ENH001, lot# 101614) overnight at 4C in binding buffer (10 mM sodium phosphate, 140 mM sodium chloride, 0.05% Triton X-100 in TE). As a negative control for DRIP, digested DNA was treated overnight with RNase H (NEB, M0297S) and then precipitated for DRIP. Dynabeads were added the next day for 2 hours. Bound Dynabeads were then washed with binding buffer three times at room temperature. DNA was eluted, phenol-chloroform extracted, and ethanol precipitated. DRIP DNA was sonicated using Covaris (Model S220) before library preparation. ChIP-seq: Cells were crosslinked with 1% formaldehyde at room temperature for 10 min, and the reaction was terminated with 125 mM Glycine at room temperature for 5 min. The crosslinking reagent was removed by spinning at 1,000 g at 4C for 5 min, and cells were washed with cold PBS three times. From this step on to ChIP elution, all buffers were prepared with freshly added cocktail of phosphatase and protease inhibitors (10 mM sodium fluoride, 10 mM sodium pyrophosphate tetrabasic, 2 mM sodium orthovanadate, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin and 1 mM PMSF). Cells were lysed on ice for 10 min using lysis buffer (5 mM HEPES, pH 7.9, 85 mM KCl, 0.5% Triton-X-100). Supernatant was removed after spinning at 1600 g at 4C for 5 min, and pellet was resuspended with nuclei lysis buffer (50 mM Tris–HCl, pH 8.0, 10 mM EDTA, 1% (wt/vol) SDS). Chromosomal DNA was sonicated using a probe sonicator on ice, then centrifuged at 14,000 g for 15 min and the supernatant was saved. Antibodies used for ChIP include: anti-Pol II (Abcam ab817, lot# GR299078-2) and anti-NELF-A which was described previously (J Cell Biochem. 107, 131-139). Sonicated DNA was incubated with antibody at 4C overnight. Dynabeads Protein A (Thermo Fisher Scientific, 10002D) was added the following day and incubated for 4 hours. After incubation, Dynabeads was washed twice in TE Sarcosyl buffer (50 mM Tris-HCl pH 8.0, 2 mM EDTA, 0.2% sarcosyl), twice in TSE1 buffer (150 mM sodium chloride, 20 mM Tris-HCl pH 8.0, 2 mM EDTA, 0.1% (wt/vol) SDS, 1% Triton-X-100), twice in TSE2 buffer (500 mM sodium chloride, 20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 0.1% (wt/vol) SDS, 0.1% Triton-X-100), twice in TSE3 buffer (250 mM lithium chloride, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% sodium deoxycholate, 1% NP-40), and twice in TE buffer (50 mM Tris-HCl pH 8.0, 2 mM EDTA). Samples were subsequently eluted and reverse-crosslinked and ethanol-precipitated. DRIP-seq and ChIP-seq libraries were built following the instruction of MicroPlex library preparation kit (Diagenode, C05010011). A total of 15 cycles of PCR amplification was performed. After amplification, libraries were purified using Agencourt AMPure XP system (Beckman Coulter, A63880) following the product manual. Quantity of the libraries was measured with Qubit dsDNA HS Assay Kit (Life Technologies, Q32851), and quality of the libraries was verified using Bioanalyzer 2100. Libraries were pooled based on index sequences.